Gene originating in human chondrocyte

ABSTRACT

This invention provides a gene specifically expressed in differentiated human chrondrocytes. By culturing the chrondrocytes in the presence of dibutyryl cAMP, the chrondrocytes are cultured in a differentiated state and any gene is searched for which has a distinction in expression between differentiated chrondrocytes and dedifferentiated chrondrocytes. This allows the gene specifically expressed in the former to be obtained.

TECHNICAL FIELD

This invention relates to a gene expressed specifically indifferentiated chondrocytes originating in human (or humanchondrocytes), a protein encoded by the gene, an antibody capable ofbinding to the protein, a method for culturing human chondrocytes in adifferentiated state, and human chondrocytes that have been cultured bythe method.

BACKGROUND ART

Searching for genes expressed specifically in chondrocytes in adifferentiated state and analysis of the properties of the chondrocytesare not only important in analyzing the mechanism of differentiation anddegeneration of cartilage, but also are indispensable for developinggene therapy for osteoarthritis and rheumatoid arthritis.

However, any method for monolayer culturing human chondrocytes in adifferentiated state has not yet been established, although culturesystems for rabbit or chicken chondrocytes in a differentiated statehave been developed. (Kato et al. Proc. Natl. Acad. Sci. USA 85,9552-9556 (1988); Oakes et al. J. Embryol. Exp. Morphol. 38, 239-263(1977).) It is recognized that human chondrocytes maintain theirdifferentiated phenotype in agarose gel (Benya P. D. and Shaffer J. D.,Cell 30, 215-224 (1982)), but that they easily lose the differentiatedphenotype in the monolayer culture which facilitates handling of cells.Accordingly, it is difficult to search for genes that are expressedspecifically in human chondrocytes in a differentiated state thereof;there has not been provided any cell culture system useful in analyzingthe properties of the chondrocytes in a differentiated state thereof.

DISCLOSURE OF INVENTION

A principle object of this invention is to establish a method ofmonolayer culture for human chondrocytes in a differentiated state andfurther to obtain a gene expressed specifically in the chondrocytes in adifferentiated state thereof.

The present inventors found that chondrocytes could be cultured in adifferentiated state by culturing the chondrocytes in the presence of acertain compound; and in addition, they searched for genes having adistinction in expression between differentiated chondrocytes anddedifferentiated chondrocytes and discovered those which werespecifically expressed in the former. Thus, this invention has beenaccomplished.

Particularly, this invention provides a DNA encoding a protein definedin (a) or (b) as described below: the DNA may be referred to as “DNA ofthis (the) invention” hereinbelow.

(a) A protein comprising an amino acid sequence set forth in SEQ ID NO:2.

(b) A protein comprising an amino acid sequence derived from the aminoacid sequence set forth in SEQ ID NO: 2 by deletion or substitution ofone or more amino acids therefrom, or by addition of one or more aminoacids thereto and capable of binding to nucleotide sequence CANNTGand/or nucleotide sequence CACNAG upon formation of a dimer,

-   -   wherein the amino acid sequence of a part of said protein        corresponding to an amino acid sequence of from amino acid no.        51 to amino acid no. 108 in SEQ ID NO: 2 is provided with not        less than 85% of homology to the amino acid sequence of from        amino acid no. 51 to amino acid no. 108 in SEQ ID NO: 2.        Preferably, DNA of this invention is a DNA defined in the        following (c) or (d):

(c) A DNA comprising a nucleotide sequence of from nucleotide no. 207 tonucleotide no. 1442 of the nucleotide sequence set forth in SEQ ID NO: 1or a complementary nucleotide sequence thereto.

(d) A DNA capable of hybridizing to the DNA defined in (c) understringent conditions.

This invention also provides a protein encoded by DNA of the invention,as well as an antibody capable of binding to the protein.

Further, the invention provides a method for culturing humanchondrocytes, which comprises monolayer culturing the chondrocytes inthe presence of a membrane-permeable cAMP analog in an amount sufficientto cause the chondrocytes to maintain a differentiated state thereof ascartilage: the method may be referred to as “the culturing method ofthis (the) invention” hereinbelow. Preferably, the membrane-permeablecAMP analog is dibutyryl cAMP (which may be denoted “dbcAMP”hereinbelow).

Still further, the invention provides human chondrocytes that have beencultured by the culturing method of the invention and that possess theproperties defined in the following (1)-(3):

-   -   (1) Exhibit a spherical shape and are abundant in extracellular        matrix;    -   (2) Can be stained with toluidine blue satisfactorily ; and    -   (3) DNA of the invention is expressed therein.

The DNA of this invention is believed to encode a novel transcriptionfactor of the basic helix-loop-helix type (bHLH), and is predicted toplay an important role such as the regulation of expression of variousgenes in the differentiation of cartilage. Therefore, a DNA of thisinvention, a protein encoded by the DNA, and an antibody capable ofbinding to the protein are useful in the analysis of mechanism of thedifferentiation and degeneration of cartilage, as well as in thedevelopment of gene therapy for osteoarthritis and rheumatoid arthritis.

According to the culturing method of this invention, chondrocytes can bemonolayer cultured in a good differentiated state, which makes it easyto search for genes having a distinction in expression betweenchondrocytes in a differentiated state thereof and chondrocytes in adedifferentiated state thereof: namely, to search for genes expressedspecifically in chondrocytes in a differentiated state thereof. Also, ananalysis of the properties of chondrocytes in a differentiated statethereof will be facilitated.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows a comparison between DEC1 and other bHLH factors in thebHLH region.

FIG. 2A is a photomicrograph showing the morphology of humanchondrocytes in monolayer culture (a-no addition of dbcAMP, b-additionof dbcAMP).

FIG. 2B is a photograph showing the morphology of human chondrocytes inmonolayer culture (the morphology of an organism) that were stained withtoluidine blue (a-no addition of dbcAMP, b-addition of dbcAMP).

FIG. 3 is a graph showing that DEC1 mRNA was induced in a fibroblaststrain, MRC5, originating in the human lung after addition of dbcAMP.

FIG. 4 is a graph showing that DEC1 mRNA was induced in Hela cellsoriginating in human uterine cancer after addition of dbcAMP.

FIG. 5 is a graph showing that EC1 mRNA was induced in rabbitchondrocytes after addition of PTH.

FIG. 6 is a graph showing that DEC1 mRNA was induced in a rabbitchondrocyte culture system after addition of dbcAMP.

FIG. 7 is a graph showing that DEC1 mRNA was induced in a renal cellstrain after addition of dbcAMP.

BEST MODE FOR CARRYING OUT THE INVENTION

Embodiments of this invention will be explained hereinbelow.

As will be later shown in the Examples, any genes expressed specificallyin chondrocytes in a differentiated state were searched for; and as aresult, the amino acid sequence set forth in SEQ ID NO: 2 has beenrevealed for the first time. As used in the present specification, theprotein having this amino acid sequence may be referred to as “DEC1.”

Through a homology search using a protein database, it was found thatDEC1 has a basic helix-loop-helix (bHLH) region (amino acid nos. 51-108in SEQ ID NO: 2). It is known that a bLHL protein forms a dimer andbinds to E box (CANNTG).

Particularly, in this bHLH region, rat HES1 (47.5%), rat HES2 (42.6%),rat HES3 (40.3%), rat HES5 (37.7%), Drosophila Hairy (abbreviated as“hairy”) (39.3%), and Enhancer of Split m7 (abbreviated as “E(spl)m7”)(37.7%) showed high homology: numerical values in the parenthesesrepresent homology levels. FIG. 1 shows corresponding bHLH regions ofthe respective proteins for comparison. The conserved residues areenclosed by the frames.

The HES family, hairy, and E(spl)m7 function as negative regulatorswhich repress transcription by binding to N box (CACNAG). (Sasai et al.Genes & Dev. 6, 2620-2634 (1992); Ishibashi et al. Eur. J. Biochem. 215,645-652 (1993); Akazawa et al. J. Biol. Chem. 267, 21879-21885 (1992);Ohsako et al. Genes & Dev. 8, 2743-2755 (1994); Dawson et al. Mol. Cell.Biol. 15, 6923-6931 (1995); Jan et al. Cell 75, 827-830 (1993).) Theyalso have the Trp-Arg-Pro-Trp (WRPW) domain (SEQ ID NO: 3) at theirC-terminus: the domain is believed to lead to suppression of a certainactivator by a repressor (Dawson et al. Mol. Cell. Biol. 15, 6923-6931(1995)). Although DEC1 resembles a bHLH factor, it does not have thisWRPW domain. Thus, DEC1 is believed to be a novel transcription factorthat modulates chondrogenesis. In view of the foregoing, it is thoughtthat DEC1 is able to bind to E box, as well as to N box.

Accordingly, the DNA of this invention encompasses: a DNA that encodes aprotein comprising the amino acid sequence set forth in SEQ ID NO: 2;and in addition, a protein comprising an amino acid sequence derivedfrom the amino acid sequence set forth in SEQ ID NO: 2 by deletion orsubstitution of one or more amino acids therefrom, or by addition of oneor more amino acids thereto and capable of binding to nucleotidesequence CANNTG and/or nucleotide sequence CACNAG (preferably,nucleotide sequence CANNTG) upon formation of a dimer,

-   -   wherein the amino acid sequence of a part of said protein        corresponding to an amino acid sequence of from amino acid no.        51 to amino acid no. 108 in SEQ ID NO: 2 is provided with not        less than 85% (preferably, not less than 90%) of homology to the        amino acid sequence of from amino acid no. 51 to amino acid no.        108 in SEQ ID NO: 2. Here, “N” in the above-described sequence        represents A, G, C, or T. For an example of nucleotide sequence        CANNTG, mentioned are CACGTG, CAGGTG, CAGTTG, and CACCTG. The        expression, “encoding a protein,” means that either one of        complementary double strands has a nucleotide sequence encoding        the protein when the DNA is double-stranded.

Substitution, deletion, or insertion of (an) amino acid residue(s) canbe generated by introducing into a nucleotide sequence, variation suchas the substitution, deletion, or insertion of nucleotide, according toa known method (e.g., site-specific mutation). Methods for determiningthe activity which allow the binding to nucleotide sequence CANNTG orCACNAG when the dimer is formed are known: for example, see Ohsako etal. Genes & Dev. 8, 2743-2755 (1994). One skilled in the art can readilyselect such substitution, deletion, or insertion of one or more aminoacid residues that would not substantially impair this activity.

Concrete examples of DNA of this invention encompass DNAs defined in thefollowing (c) and (d):

(c) A DNA comprising a nucleotide sequence of from nucleotide no. 207 tonucleotide no. 1442 of the nucleotide sequence set forth in SEQ ID NO: 1or a complementary nucleotide sequence thereto.

(d) A DNA capable of hybridization to the DNA defined in (c) understringent conditions.

Here, the “stringent conditions” means the conditions under which aso-called specific hybrid is formed, but any non-specific hybrid is notformed. These conditions may be difficult to be accurately expressed asnumerical values; and for example, mentioned is a temperature in therange of from Tm to Tm minus 20° C. where the Tm is the one for aperfectly matched hybrid, such as that between highly homologous nucleicacids, or alternatively the conditions under which DNAs having homologyof not less than 80% hybridize to each other, but nucleic acids havinghomology lower than that do not hybridize to each other.

The DNA. of this invention is preferably one that encodes the amino acidset forth in SEQ ID NO: 2, and is more preferably the DNA of (c)described above.

As will be later shown in the Examples, one of the nucleotide sequencesfor DNA of the invention has been determined; therefore, it is possibleto synthesize the DNA based on this sequence. The DNA can also beobtained from chromosomal DNAs by PCR or hybridization usingoligonucleotides or probes that have been prepared based on this verynucleotide sequence. Alternatively, the DNA can further be obtainedeither by carrying out RT-PCR with cartilage mRNA or by screening a cDNAlibrary, such as cartilage, with polynucleotides having a nucleotidesequence that encodes the whole or a part of DEC1 as probes.

The protein encoded by DNA of this invention is a protein defined in (e)or (f) described below: the protein may be referred to as “theprotein(s) of this (the) invention” hereinbelow.

(e) A protein comprising an amino acid sequence set forth in SEQ ID NO:2.

(f) A protein comprising an amino acid sequence derived from the aminoacid sequence set forth in SEQ ID NO: 2 by deletion or substitution ofone or more amino acids therefrom, or by addition of one or more aminoacids thereto and capable of binding to nucleotide sequence CANNTGand/or nucleotide sequence CACNAG upon formation of a dimer, wherein theamino acid sequence of a part of said protein corresponding to an aminoacid sequence of from amino acid no. 51 to amino acid no. 108 in SEQ IDNO: 2 is provided with not less than 85% of homology to the amino acidsequence of from amino acid no. 51 to amino acid no. 108 in SEQ ID NO:2.

The protein of this invention can be produced by the following steps: aDNA of this invention is inserted into a known expression vector toconstruct a recombinant plasmid; transformed cells are obtained byintroducing this recombinant plasmid thereto; the transformed cells arecultured in a suitable medium to allow a protein of the invention toform and accumulate in the culture; and said protein is harvested fromthe culture.

Host-vector systems ordinarily used to express exogenous proteins can beemployed as cell and expression vectors. For example, mentioned are acombination of a prokaryotic cell such as E. coli and an expressionvector adapted to the cell and a combination of a eukaryotic cell suchas a mammalian cell and an expression vector adapted to the cell.Culture media and culturing conditions may appropriately be selected inaccord with the cells to be employed.

The proteins of this invention may be expressed as fusion proteins withother proteins. Also, the proteins of the invention may be expressed intheir full-length, or alternatively, portions thereof may be expressedas partial peptides.

Cultures, as used herein, are media and cells in the media. Harvestingproteins of this invention from the cultures can be carried outaccording to known protein purification methods which utilize, amongothers, the above-mentioned activity of proteins of the invention as anindex.

Antibodies capable of binding to the proteins of this invention can beprepared according to standard methods using the proteins of theinvention as antigens: the antibodies may be referred to as “antibody(ies) of this (the) invention.” The antibodies of this invention may bemonoclonal antibodies or polyclonal antibodies.

The protein of this invention may be used intact as an antigen; however,it is preferred that the protein be conjugated to keyhole lymphethemocyanine, bovine serum albumin, egg white albumin, etc. and/or becombined with adjuvants for use as the antigen.

An animal to be immunized such as a mouse, rabbit, guinea pig, or sheepis immunized by administration of the antigen described above viapercutaneous, intraperitoneal, or intravenous injections or the like. Apolyclonal antibody can, for example, be obtained by collecting serumfrom the immunized animal.

A monoclonal antibody can, for example, be obtained in the followingmanner. After an animal to be immunized such as a mouse, rabbit, guineapig, or sheep is immunized by administration of the antigen describedabove via percutaneous, intraperitoneal, or intravenous injections orthe like, its spleen or lymph node is extracted. Cells taken from thisare fused with myeloma cells, which are preferably derived from the sameanimal species as that of the immunized animal, to create a hybridoma. Acell strain is selected by repeating screening and cloning from theobtained hybridoma: the strain incessantly produces an antibody specificto the above-mentioned antigen. A monoclonal antibody is produced in asuitable medium by culturing the thus-selected cell strain in themedium; or alternatively, it is produced in an ascite fluid or the likeby culturing the strain in vivo, such as mouse abdominal cavities.

Purification methods for the resulting polyclonal and monoclonalantibodies include salting out with ammonium sulfate, ion-exchangechromatography using a DEAE cellulose column or the like, affinitychromatography using a Protein A column, and immunoabsorptionchromatography. The antibodies of this invention can be detected byimmunoassays using the proteins of the invention or labeled antibodies.

The antibodies of this invention may be fragmented ones, so long as theyretain antigen-binding sites (Fab). Specifically mentioned as thepresent fragmented antibody is a fragment containing Fab that can beobtained by digesting the present antibody with a protease such aspapain which does not digest the antigen-binding site.

The antibodies of this invention may be labeled by being bound tolabeling substances. The labeling substances are not particularlylimited, so long as they can ordinarily be used in labeling of proteins;and mentioned are enzymes, isotopes, fluorescent substances, etc.

Next, the culturing method of this invention will be explained. Themethod of the invention is characterized in that it comprises monolayerculture of chondrocytes in the presence of a membrane-permeable cAMPanalog in an amount sufficient to cause the chondrocytes to maintain adifferentiated state thereof as cartilage.

Monolayer culture of chondrocytes can be carried out in a manner similarto the conventional monolayer culture of chondrocytes, except that it isto be done in the presence of the membrane-permeable cAMP analog. Forexample, the media that are used for culture include α-modified Eagle'smedium containing fetal bovine serum, ascorbic acid, an antibiotic, etc.as appropriate.

The membrane-permeable cAMP analog is an analog of cAMP that has theability to permeate the membrane without impairing its function as aso-called second messenger of cAMP; preferably, it is dibutyryl cAMP.

The quantity of the membrane-permeable cAMP analog present in a mediummay be such that it is sufficient to cause the chondrocytes to maintaina differentiated state thereof as cartilage. For example, in the case ofdibutyryl cAMP, it is preferably 0.3-0.5 mM.

As used in the present specification, the “differentiated state” meansthat the chondrocytes possess at least the properties described in thefollowing (1) to (2):

-   -   (1) Exhibit a spherical shape and are abundant in extracellular        matrix; and    -   (2) Can be stained with toluidine blue satisfactorily.

The membrane-permeable cAMP analog in an amount sufficient to causechondrocytes to maintain a differentiated state thereof as cartilage canalso induce the differentiation of dedifferentiated chondrocytes.

Furthermore, this invention provides human chondrocytes that have beencultured by the culturing method of the invention and that possess theproperties described in the following (1) to (3):

-   -   (1) Exhibit a spherical shape and are abundant in extracellular        matrix;    -   (2) Can be stained with toluidine blue satisfactorily; and    -   (3) DNA of this invention is expressed therein.

Since toluidine blue selectively stains sulfated proteoglycan, thechondrocytes of this invention synthesize the sulfated proteoglycan.

These chondrocytes express mRNAs for collagen of Type I and Type II aswell as those for aggrecan.

EXAMPLES

This invention will be explained by way of examples hereinbelow.

Example 1 Culture of Chondrocytes in Differentiated State

Epiphyseal cartilage of the femur knee joint of a human fetus that wasnaturally aborted about 25 weeks of pregnancy (obtained from NormanBethune University of Medical Sciences, Department of Pathology) wasobtained. Chondrocytes were isolated from this cartilage according tothe same method as that described in Shimomura et al. Calcif. TissueRes. 19, 179-187 (1975), except that the finely cut cartilage wasincubated in a α-modified Eagle's medium (α-MEM) containing 3 mg/mlcollagenase (Type IA, Sigma) for 3 h. The cells were seeded at 1×105cells per Type I collagen-coated dish and maintained in α-MEM (10ml/dish) containing 10% fetal bovine serum, 50 μg/ml of ascorbic acid,32 unit/ml of penicillin, and 40 μg/ml of streptomycin. Dibutyryl cAMP(dbcAMP) (1 mM) was added to the culture medium when the cells becamesubconfluent. Cells were cultured over two days either in the presenceor in the absence of dbcAMP. Then, while the cells were harvested, themorphological change of cells was examined. After fixing with ethanol,the cells were stained with toluidine blue.

The chondrocytes cultured in the presence of dbcAMP exhibited aspherical shape and were abundant in extracellular matrix, whereas thechondrocytes cultured in its absence were fibroblast-like and have aspindle shape, and were deficient in extracellular matrix. FIG. 2A showsa photomicrograph of the morphology of cells on the 6th day afteraddition of dbcAMP (a-no addition of dbcAMP, b-addition of dbcAMP).

In the staining with toluidine blue that should selectively stainsulfated proteoglycan, the chondrocytes cultured in the presence ofdbcAMP were stained satisfactorily, whereas the chondrocytes cultured inits absence were hardly stained. FIG. 2B shows the results of stainingwith toluidine blue of cells on the 12th day after addition of dbcAMP(a: no addition of dbcAMP, b: addition of dbcAMP).

Expression of the mRNA for Type I and Type II collagen, and aggrecan,which serve as molecular markers, was investigated by RT-PCR.Expressions of the molecular markers were compared in the presence ofdbcAMP as well as in its absence; as a result, it was indicated that thedifferentiated state was maintained in the presence of dbcAMP.

Accordingly, it was recognized that the chondrocytes cultured in thepresence of dbcAMP maintained a differentiated state thereof ascartilage (namely, the maintenance of a differentiated phenotype).

When dose-dependence of the above-noted effect by dbcAMP was studied,said effect increased in a dose-dependent manner, reaching its maximumat 0.3-0.5 mM.

It has been reported that bFGF and TGF-β stabilize or stimulate theexpression of rabbit or chicken chondrocytes of the differentiatedphenotype. Employing bFGF (0.4 ng/ml) and TGF-β (3 ng/ml), thechondrocytes were cultured similarly to above, but they did not maintaintheir differentiated phenotype in human chondrocytes.

Exampl 2 Expression of Specific Genes in the Differentiated Chondrocytes

Total RNA was extracted from the chondrocytes cultured in the presenceof dbcAMP and those cultured in the absence of dbcAMP (as described inExample 1) by the guanidinethiocyanate/cesium trifluoroacetate method.Poly(A)+RNA was concentrated using Oligotex-dT30 (Roche). Thesubtractive hybridization was used to select clones in which mRNA wasobserved that had been expressed in the differentiated chondrocytes(+dbcAMP) but not in the dedifferentiated chondrocytes (−dbcAMP). Usinga PCR select cDNA subtraction kit (Clonetech), cDNA synthesized from themRNA of the differentiated chondrocytes was allowed to hybridize with anexcess amount of cDNA of from the mRNA of the dedifferentiatedchondrocytes. The cDNA that did not hybridize, namely that was expressedin a differentiated state was amplified by suppression PCR according tothe manufacturer's manual. The resulting PCR product was cloned intopGEM-T (Promega), a T tail vector, and the nucleotide sequencedetermination was carried out on about 120 clones. One clone (pSUB37)was selected for further analysis, and the corresponding protein productwas named DEC1.

NcoI-PstI fragment from pSUB37 was used as a probe to study theexpression of DEC1 mRNA in various human fetus tissues by Northern blotanalysis. Consequently, DEC1 was expressed in the cartilage, spleen,intestine, and lung; and it was also expressed in the heart, liver,brain, and stomach, although in small amounts. The Northern blotanalysis was conducted in the following manner. Total RNA samples (5 or10 μg) were electrophoresed on a 1% agarose gel containing formaldehydeand were transferred to Highbond-N-membranes (Amersham). Total RNAsamples of various human fetus tissues were provided by Dr. Li Yu atNorman Bethune University of Medical Sciences, which were intended forthe study of tissue distribution. NcoI-PstI fragment from pSUB37 waslabeled with [³²P]dCTP, and it was used as a hybridization probe. Themembranes were washed with 2×SSC containing 0.5% SDS at 65° C. for 30min. Biomax X-ray films were exposed to the washed membranes usingsensitizing films at −70° C.

The full-length nucleotide sequence of DEC1 cDNA was determined in thefollowing manner. A full-length cDNA of DEC1 was isolated by RACE (rapidamplification cDNA ends method) using a Marathon cDNA amplification kit(Clonetech). Specifically, a double-stranded cDNA was ligated to aMarathon cDNA adapter and subjected to suppression PCR. Reaction wascarried out using an adapter primer and a gene-specific primer that hadbeen designed for DEC 1 based on the nucleotide sequence of pSUB37. Theamplified cDNA sample was separated on a 4% polyacrylamide gel, DNA ofthe main band was extracted from the gel, and it was subcloned intopGEM-T. Double-stranded DNA of the subcloned plasmid and a series ofsynthetic oligonucleotides were used as a sequence-determining templateand as a specific primer, respectively. DNA sequence determination wascarried out by the Sanger method using either a sequenase7-deaza-dGTPDNA sequencing kit (Amersham) or an ABI PRIZM 310autosequencer (Perkin-Elmer).

The nucleotide sequence of DEC1 cDNA thus determined and the amino acidsequence deduced therefrom are set forth in SEQ ID NO: 1. This aminoacid sequence alone is also set forth in SEQ ID NO: 2. DEC1 cDNA has anopen reading frame of 1236 bp. A 2922 bp length, excluding the poly Aregion, is well in accord with the size of mRNA (3.1 kb) that wasobtained by the Northern blot analysis described above. Since there is astop codon in the 5′-region which serves as an inframe, the first ATG isregarded as an initiation codon. The sequence around the first ATGcoincides with a Kozak consensus sequence (GCCGCCA/GCCATGG). Thus, DEC1comprises 412 amino acids and its calculated molecular weight is 45.5kDa.

Example 3

(1) Materials and Methods

Chondrocytes were isolated from the rib growth plate and restingcartilage of ribs of a male Japanese white rabbit (four-weeks old)according to the method as already reported in Shimomuraet al. Calcif.Tissue Res. 19, 179-187 (1975).

These cells were seeded at 5×10⁵ cells per 10 mm plastic culture dishand maintained in 10 ml of α-MEN supplemented with 10% FBS, 60 mg/ml ofkanamycins, 250 ng/ml of amphotericin B, and 50 unit/ml of penicillin Gat 37° C. in the air containing 5% CO₂. After cells reached confluent,the cells were washed with PBS, transferred to a fresh α-MEN (10 ml)containing no serum, and maintained for 48 h. From 1 to 24 h before thecompletion of incubation, 1 mM dbcAMP or 10×x⁻⁷ M human recombinantPTH-(1-84) was added to the medium.

Human embryonic pulmonary fibroblasts (MRC-5), human cervix uteriepithelial cells (Hela), human hepatoma cells (HepG2) and canine renalepithelial cells (MDCK) were obtained from the Gene Bank of TheInstitute of Physical and Chemical Research. Cells were cultured inmodified Dalbeco Eagle's media (DMEM) supplemented with 10% FBS untilthey reached confluent. After the cultures had turned confluent, thecells were washed with 10% PBS, transferred to fresh DMEM (10 ml)containing no serum, and maintained for 48 h. From 1 to 24 h before thecompletion of incubation, 1 mM dbcAMP was added to the media. Then,cells were harvested for RNA preparations.

(2) Northern Blot Analysis

Total RNAs were extracted from the cultured cells by theguanidine•thiocyanate/cesium trifluoroacetate method. (Smale G. andSasse J., Anal. Biochem. 203, 352-356 (1992).) The total RNA samples(8-20 μg) were electrophoresed on a 1% agarose gel containingformaldehyde and were transferred to NYTRAN membranes (Schleicher &Schuell, Japan). A 1.1 kb NcoI-PstI fragment from pSUB37 was labeledwith [³²P]dCTP, and it was used as a hybridization probe. The membraneswere washed with 0.2×SSC containing 0.5% DSD at 55° C. for 30 min.BioMax X-ray films (Eastman Kodak Co., Rochester, N.Y.) were exposed tothe washed membranes using sensitizing screens at −70° C.

(3) Results

(I) Inhuman pulmonary fibroblast cell line MRC5, DEC1 mRNA was inducedfrom 1 to 24 h after addition of dbcAMP (FIG. 3).

(II) In the Hela cells originating in human uterine cancer, DEC1 mRNAwas also induced within 1 h after addition of dbcAMP (FIG. 4).

(III) In the rabbit chondrocytes, DEC1 mRNA was induced from 1 to 24 hafter addition of PTH (FIG. 5).

(IV) In the rabbit chondrocyte culture system, it was also confirmedthat DEC1 mRNA had been induced after addition of dbcAMP (FIG. 6)

(v) In the HepG2 cell strain originating in human hepatic cell line, nochange in the level of DEC1 mRNA was observed between 1 to 6 h afteraddition of dbcAMP.

(VI) In the renal cell strain, the addition of dbcAMP also induced DEC1mRNA (FIG. 7).

The above results have indicated that DEC1bHLH transcription factor isinvolved in the mechanism of action of PTH/PTH-rp in chondrocytes.Furthermore, the DEC1bHLH transcription factor was induced in responseto the cAMP within one hour in almost all the mesenchymal and epithelialcells tested. This has suggested that the present transcription factoralmost universally participates in the gene expression of the cAMPsignal system.

INDUSTRIAL APPLICABILITY

As has been explained above, according to this invention there areprovided a gene specifically expressed in differentiated humanchrondrocytes, a method for culturing human chrondrocytes in adifferentiated state, and human chrondrocytes that have been cultured bythe method. These are important not only in the analysis ofdifferentiation and degeneration of cartilage, but also in thedevelopment of gene therapy for osteoarthritis and rheumatoid arthritis.Moreover, they are believed to be useful in the treatment of diseasesinvolving other cAMP systems, because cAMP induces DEC1 mRNA in a largenumber of cells other than the chrondrocytes.

1-7. (canceled)
 8. A method of detecting a differentiated chondrocytecomprising: contacting a chondrocyte with an anti-DEC1 antibody orantigen binding fragment thereof capable of specifically binding to aDEC-1 protein comprising the amino acid sequence of SEQ ID NO:
 2. 9. Themethod of claim 8, wherein said antibody or antigen binding fragmentthereof is selected from the group consisting of polyclonal antibodies,monoclonal antibodies and FAB fragments.
 10. The method of claim 8,wherein said antibody or antigen binding fragment thereof is labeled.11. The method of claim 10, wherein said label is selected from thegroup consisting of enzymes, isotopes and fluorescent substances.
 12. Amethod for modulating chondrogenesis, using an anti-DEC1 antibodycapable of binding to the DEC1 protein comprising the amino acidsequence of SEQ ID NO:
 2. 13. A method for diagnosing abnormalities inchondrogenesis, using an anti-DEC1 antibody capable of binding to theDEC1 protein comprising the amino acid sequence of SEQ ID No: 2: